Combination therapy for eradicating detectable HCV-RNA in patients having chronic hepatitis C infection

ABSTRACT

There is disclosed a method for treating a patient having chronic hepatitis C infection to eradicate detectable HCV-RNA involving a combination therapy using a therapeutically effective amount of ribavirin and a therapeutically effective amount of interferon-alpha for a time period of from 20 up to 80 weeks.

Chronic infection with hepatitis C virus is an insidious andslow-progressing disease having a significant impact on the quality oflife. It can eventually result in cirrhosis of the liver, decompensatedliver disease and/or hepatocelluar carcinoma.

Alpha interferon monotherapy is commonly used to treat chronic hepatitisC infection. However, this treatment is not always effective andsometimes results in intolerable side effects related to the dosage andduration of therapy. Ribavirin has been proposed as a monotherapytreatment for chronic hepatitis C infection (Thomas et al. AASLDAbstracts, Hepatology Vol. 20, NO. 4, Pt 2, Number 440, 1994). However,this monotherapy treatment has usually been found relatively ineffectiveand has its own undesirable side effects. Combination therapy of alphainterferon and ribavirin has been proposed (Lai, et al. Symposium to the9th Biennial Scientific Meeting Asian Pacific Association for the Studyof the Liver. 1994). Preliminary results suggest that the combinationtherapy may be more effective than either monotherapy. Hayden F G,Schlepushkin A N. Combined interferon-a2, rimantadine hydrochloride, andribavirin inhibition of influenza virus replication in vitro. AntimicrobAgents Chemother. 1984;25:53-57. Schvarcz R, Ando Y, S{grave over ()}nnerborg A, Weiland O. Combination treatment with interferon alfa-2band ribavirin for chronic hepatitis C in patients who have failed toachieve sustained response to interferon alone: Swedish experience. JHepatology. 1995;232 (Suppl 2):17-21. Brouwer J T, Nevens F, MichielsenP, et al. What options are left when hepatitis C does not respond tointerferon? Placebo-controlled Benelux multicentre retreatment trial onribavirin monotherapy versus combination with interferon. J Hepatol.1994;212 (Suppl 1):S17. Abstract WP2/08. Chemello L, Cavalletto L,Bemardinello E, et al. Response to ribavirin, to interferon and to acombination of both in patients with chronic hepatitis C and itsrelation to HCV genotypes. J Hepatol. 1994;212 (Suppl 1):S12. AbstractGS5/29. However, no one has described methods using alpha interferon andribavirin which eradicate HCV-RNA in any long-term, effective manner.

There is a definite need for a method for treating chronic hepatitis Cinfection with a combination of alpha interferon and ribavirin whicheradicates HCV-RNA in any long-term, effective manner.

SUMMARY OF THE INVENTION

The present invention involves a method of treating a patient havingchronic hepatitis C infection to eradicate detectable HCV-RNA comprisingadministering a therapeutically effective amount of ribavirin and atherapeutically effective amount of interferon-alpha for a time periodof 20 to 30 weeks, such that at least about 30% of the patients havingno detectable HCV-RNA at the end of said 20 to 30 week period also haveno detectable HCV-RNA for at least 24 weeks after the end of saidadministration. Preferably, at least about 40% of the patients having nodetectable HCV-RNA at the end of said 20 to 30 week period also have nodetectable HCV-RNA for at least 24 weeks after the end of saidadministration.

In another embodiment the present invention relates to a method oftreating a patient having chronic hepatitis C infection to eradicatedetectable HCV-RNA comprising administering a therapeutically effectiveamount of ribavirin and a therapeutically effective amount ofinterferon-alpha for a time period of 40 to 50 weeks, such that at leastabout 40% of the patients having no detectable HCV-RNA at the end ofsaid 40 to 50 week period also have no detectable HCV-RNA for at least24 weeks after the end of said administration. Preferably, at leastabout 50% of the patients having no detectable HCV-RNA at the end ofsaid 40 to 50 week period also have no detectable HCV-RNA for at least24 weeks after the end of said administration.

Another embodiment of the invention relates to a method of treating apatient having chronic hepatitis C infection to eradicate detectableHCV-RNA comprising administering a therapeutically effective amount ofribavirin and a therapeutically effective amount of interferon-alpha fora time period of 60 to 80 weeks, such that at least about 50% of thepatients having no detectable HCV-RNA at the end of said 60 to 80 weekperiod also have no detectable HCV-RNA for at least 24 weeks after theend of said administration. Preferably, at least about 60% of thepatients having no detectable HCV-RNA at the end of said 60 to 80 weekperiod also have no detectable HCV-RNA for at least 24 weeks after theend of said administration.

One aspect of the invention involves a method of treating a patienthaving chronic hepatitis C infection having HCV genotype other than type1 and having a viral load of less than or equal to 2 million copies perml of serum as measured by HCV-RNA quantitative PCR to eradicatedetectable HCV-RNA comprising administering a therapeutically effectiveamount of ribavirin and a therapeutically effective amount ofinterferon-alpha for a time period of 20 to 30 weeks, such that at leastabout 70% of the patients having no detectable HCV-RNA at the end ofsaid 20 to 30 week period also have no detectable HCV-RNA for at least24 weeks after the end of said administration. Preferably, at leastabout 80% of the patients having no detectable HCV-RNA at the end ofsaid 20 to 30 week period also have no detectable HCV-RNA for at least24 weeks after the end of said administration.

Another aspect of the invention relates to a method of treating apatient having chronic hepatitis C infection having HCV genotype otherthan type 1 and having a viral load of greater than 2 million copies asmeasured by HCV-RNA/qPCR to eradicate detectable HCV-RNA comprisingadministering a therapeutically effective amount of ribavirin and atherapeutically effective amount of interferon-alpha for a time periodof 20 to 30 weeks, such that at least about 50% of the patients havingno detectable HCV-RNA at the end of said 20 to 30 week period also haveno detectable HCV-RNA for at least 24 weeks after the end of saidadministration. Preferably, at least about 60% of the patients having nodetectable HCV-RNA at the end of said 20 to 30 week period also have nodetectable HCV-RNA for at least 24 weeks after the end of saidadministration.

Yet another aspect of the invention involves a method of treating apatient having chronic hepatitis C infection having HCV genotype type 1and having a viral load of less than or equal to 2 million copies asmeasured by HCV-RNA/qPCR to eradicate detectable HCV-RNA comprisingadministering a therapeutically effective amount of ribavirin and atherapeutically effective amount of interferon-alpha for a time periodof 20 to 30 weeks, such that at least about 30% of the patients havingno detectable HCV-RNA at the end of said 20 to 30 week period also haveno detectable HCV-RNA for at least 24 weeks after the end of saidadministration. Preferably, at least about 40% of the patients having nodetectable HCV-RNA at the end of said 20 to 30 week period also have nodetectable HCV-RNA for at least 24 weeks after the end of saidadministration.

Still another embodiment of the invention relates to a method oftreating a patient having chronic hepatitis C infection having HCVgenotype type 1 and having a viral load of greater than 2 million copiesas measured by HCV-RNAqPCR to eradicate detectable HCV-RNA comprisingadministering a therapeutically effective amount of ribavirin and atherapeutically effective amount of interferon-alpha for a time periodof 20 to 30 weeks, such that at least about 15% of the patients havingno detectable HCV-RNA at the end of said 20 to 30 week period also haveno detectable HCV-RNA for at least 24 weeks after the end of saidadministration. Preferably, at least about 20% of the patients having nodetectable HCV-RNA at the end of said 20 to 30 week period also have nodetectable HCV-RNA for at least 24 weeks after the end of saidadministration.

Preferably, the amount of ribavirin administered is from 400 to 1200 mgper day. More preferably, the amount of ribavirin administered is from800 to 1200 mg per day.

The interferon-alpha administered is preferably selected from interferonalpha-2a, interferon alpha-2b, a consensus interferon, a purifiedinterferon alpha product or a pegylated interferon-alpha. Morepreferably, the interferon-alpha is selected from interferon alpha-2a,interferon alpha-2b, or a purified interferon alpha product and theamount of interferon-alpha administered is from 2 to 10 million IU perweek on a weekly, TIW, QOD or daily basis. In a preferred embodiment,the interferon-alpha administered is interferon-alpha-2b and the amountof interferon-alpha is administered 3 million IU TIW.

Alternatively, the interferon-alpha administered is consensus interferonand the amount of interferon-alpha administered is from 1 to 20micrograms per week on a weekly, TIW, QOD or daily basis. In anotherembodiment, the interferon-alpha administered is a pegylated interferonalpha-2b and the amount of interferon-alpha administered is from 0.5 to2.0 micrograms/kilogram per week on a weekly, TIW, QOD or daily basis.Alternatively, the interferon-alpha administered is a pegylatedinterferon alpha-2a and the amount of interferon-alpha administered isfrom 20 to 250 micrograms/kilogram per week on a weekly, TIW, QOD ordaily basis.

The present invention has surprisingly found that, when compared tointerferon-alpha treatment alone or ribavirin alone, therapy with acombination of a therapeutically effective amount of ribavirin and atherapeutically effective amount of interferon-alpha for a time periodof at least 20 to 30 weeks results in ten times more patients having nodetectable HCV-RNA in their serum at least 24 weeks after termination oftherapy than by either monotherapy.

DETAILED DESCRIPTION

The term “interferon-alpha” as used herein means the family of highlyhomologous species-specific proteins that inhibit viral replication andcellular proliferation and modulate immune response. Typical suitablealpha interferons include, but are not limited to, recombinantinterferon alpha-2b such as Intron-A interferon available from ScheringCorporation, Kenilworth, N.J., recombinant interferon alpha-2a such asRoferon interferon available from Hoffmann-La Roche, Nutley, N.J.,recombinant interferon alpha-2C such as Berofor alpha 2 interferonavailable from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield,Conn., interferon alpha-n1, a purified blend of natural alphainterferons such as Sumiferon available from Sumitomo, Japan or asWellferon interferon alpha-n1 (INS) available from the Glaxo-WellcomeLtd., London, Great Britain, or a consensus alpha interferon such asthose described in U.S. Pat. Nos. 4,897,471 and 4,695,623 (especiallyExamples 7, 8 or 9 thereof) and the specific product available fromAmgen, Inc., Newbury Park, Calif., or interferon alpha-n3 a mixture ofnatural alpha interferons made by Interferon Sciences and available fromthe Purdue Frederick Co., Norwalk, Conn., under the Alferon Tradename.The use of interferon alpha-2a or alpha 2b is preferred. Sinceinterferon alpha 2b, among all interferons, has the broadest approvalthroughout the world for treating chronic hepatitis C infection, it ismost preferred. The manufacture of interferon alpha 2b is described inU.S. Pat. No. 4,530,901.

Ribavirin, 1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide,available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif., isdescribed in the Merck Index, compound No. 8199, Eleventh Edition. Itsmanufacture and formulation is described in U.S. Pat. No. 4,211,771.

A person suffering from chronic hepatitis C infection may exhibit one ormore of the following signs or symptoms:

-   (a) elevated ALT,-   (b) positive test for anti-HCV antibodies,-   (c) presence of HCV as demonstrated by a positive test for HCV-RNA,-   (d) clinical stigmata of chronic liver disease,-   (e) hepatocelluar damage.

To practice the invention, alpha interferon (hereinafter α-IFN) andribavirin are administered to the patient exhibiting one of more of theabove signs or symptoms in amounts sufficient to eliminate or at leastalleviate one or more of the signs or symptoms. In a preferredembodiment, the combination therapy of the invention is administered toa patient who has failed to remain HCV-RNA free after interferon-alphamonotherapy.

The ribavirin is administered to the patient in association with theα-IFN, that is, the α-IFN dose is administered during the same period oftime that the patient receives doses of ribavirin. Most α-IFNformulations are not effective when administered orally, so thepreferred method of administering the α-IFN is parenterally, preferablyby subcutaneous, IV, or IM, injection. The ribavirin may be administeredorally in capsule or tablet form in association with the parenteraladministration of α-IFN. Of course, other types of administration ofboth medicaments, as they become available are contemplated, such as bynasal spray, transdermally, by suppository, by sustained release dosageform, etc. Any form of administration will work so long as the properdosages are delivered without destroying the active ingredient.

Detectable HCV-RNA in the context of the present invention means thatthere is less than 100 copies per ml of serum of the patient as measuredby quantitative, multi-cycle reverse transcriptase PCR methodology.HCV-RNA is preferably measured in the present invention by themethodology described below. This methodology is referred to herein asHCV-RNA/qPCR RNA is extracted from patient serum using a guaninidiumthiocyanate-phenol-chloroform mister followed by ethanol-ammoniumacetate precipitation. The precipitated RNA is centrifuged and theresulting pellet is dried in a Centrivap console (Labconco, Kansas City,Mo.). The dry pellet is then resuspended in 30 microliters of an Rnasin(Promega Corp., Madison, Wis.), dithiothritol, anddiethylpyrocarbonate-treated water mixture. Samples are kept at or below−20° C. until RNA reverse transcription (RT) and PCR.

In order to convert the entire RNA sequence into cDNA in the RTreaction, random hexadeoxyribonucleotides (Pharmacia Biotech,Piscataway, N.J.) are used as primers for the first strand cDNAsynthesis. Two aliquots of 3 microliters of resuspended sample is addedto 3 microliters of 100 ng/μl random primers and denaturated at 70° C.,then reverse transcribed at 40° C. for one hour using M-MLV reversetranscriptase (USB, Cleveland, Ohio) in standard buffer containing 5 mMMgCl₂. The final RT reaction volume is 26 μl. The PCR is startedimmediately following the reverse transcription.

A modified version of the PCR method is performed using heat-stable Taqpolymerase to amplify the cDNA. Seventy-five microliters of PCR mix isadded to the entire RT reaction volume (26 μl) to a final MgCl₂concentration of 1.5 mM in a total volume of 101 μl. Each 101 μl sampleis then split into 50.5 μl, and a layer of mineral oil is placed on topto prevent evaporation.

The PCR cycle consists of annealing for 90 sec., extension for 90 sec.,and denaturation for 90 sec., at 55° X, 74° C. and 94° C., respectively.Thermocycling samples is submitted to a final 74° C. extension for 10minutes. Four different cycle sets are used. Bu ;loading the sample induplicate, and splitting these samples evenly after RT, there are fourtubes from one sample. Each of the four tubes is given a different cyclenumber, enhancing sensitivity and accuracy in the quantitation process.The thermocycling efficiency will be assessed by satisfactoryamplification of known copy number RNA standards included in each set of60 tubes. Two primer sets are used for the amplification, both from the5′ untranslated region of the HCV genome. Both of these primer sets arehighly conserved and detect all known subtypes of HCV. Primer set 1:upstream 5′-GTG GTC TGC GGA ACC GGT GAG T-3′, downstream 5′-TGC ACG GTCTAC GAG ACC TC-3′ which produced a 190 bp product. Primer set 2:upstream 5′-CTG TGA GGA ACT ACT GTC TTC-3′, downstream 5′-CCC TAT CAGGCA GTA CCA CM-3′which produced a 256 bp product.

The amplified cDNA is then electrophorised in 3% agarose gel andtransferred to nylon membrane. The target DNA is detected by Southernblotting and immunostaining using a nonradioactive digoxigenin-labeledDNA probe. These procedures are performed using automated instrumentsfor PCR thermocycling, agarose gel electrophoresis, vacuum-transferSouthem blot, hybridization, and immunostaining. Each membrane containsknown copy number serially diluted standards which are used to constructstandard curves for quantitative measurement of the specimen bands.Originally standard curves are made from carefully diluted HCV-RNA fromtranscribed clones. Radioactive incorporation studies, gelelectrophoresis, and OD 260 are performed on the transcripts todetermine that they are of the expected length. After the production ofthe RNA transcripts quantitated clone standards “pooled” standards aregenerated which better represent the heterogeneous nature of HCV, onewould encounter in natural infection. These pools are made by combininglarge amounts of serum or plasma from known infected individuals. Theserum/plasma pools are calibrated with PCR, against the clonetranscripts and then diluted in the known PCR-negative fluids. Finally,the higher copy number samples of the pools are checked against the cDNAQuantiplex nucleic acid detection system from Chiron Inc. (Emeryville,Calif.). These “double quantitated” pools are aliquoted and saved at−70° C. Dilutions of 5,000,000, 1,000,000, 500,000, 100,000, 10,000, and1000 copies/ml are used in each experiment.

Each Southern blot membrane is scanned into a computer using anautomated scanner/densitometer, at intervals during development todetermine when the standard curve is most linear. The resultantelectronic images are then measured for band area and mean band density.All of the reading are standardized to integrated band density andcompared to the standard curve to obtain a numerical value of viral copynumber for each band.

The following clinical protocols were performed:

Study 1

Overall Design and Plan of the Study

This was a prospective, multicenter, randomized, double-blind,parallel-group. The study compared treatment with INTRON® A plusribavirin to treatment with INTRON® A plus placebo for 24 weeks inpatients with compensated chronic hepatitis C who had responded to oneor two previous courses of alpha interferon (INTRON® A, Roferon®-A, orWellferon®) therapy (minimum of 3 MU to a maximum of 6 MU QOD or TIW fora minimum of 20 weeks to a maximum of 18 months) and who had relapsedafter the most recent course of alpha interferon therapy. Eligiblepatients had chronic hepatitis C confirmed by positive serum HCV-RNA,liver biopsy, and laboratory tests.

Patients were randomized to treatment with either INTRON® A plusribavirin or INTRON® A plus placebo. The dose of INTRON® A was 3 MU SCTIW; the dose of ribavirin was 1000 or 2000 mg PO daily (based onweight) in two divided doses. Treatment group assignments were made inequal ratios by a Central Randomization Center. The randomizationprocedure was designed to attempt to balance the treatment groups,within and across sites, with respect to presence or absence ofcirrhosis in the pretreatment liver biopsy, serum HCV-RNA/qPCR level,and HCV genotype.

Study treatment was administered for 24 weeks. The total course of thestudy was 48 weeks to determine long-term effect of treatment.

During treatment and posttreatment follow-up, biochemical (ALT),virological (HCV-RNA), and histological (liver biopsy) examinations wereused to assess the nature and duration of response to study treatment.The primary efficacy variable was the overall response defined as lossof serum HCV-RNA/qPCR (<100 copies/mL) as measured at 24 weeks followingthe end of therapy associated with an improvement in posttreatment liverbiopsy as measured by the Knodell Histology Activity index (HAI).Normalization of ALT was also examined as a secondary efficacy variable.The safety of the study treatments was assessed by monitoring selectedlaboratory parameters and by also recording and evaluating theoccurrence of any adverse events.

Treatment Regimens

The study treatment regimens were either:

-   INTRON® A 3 MU SC TIW plus ribavirin 1000 or 1200 mg/day PO in two    divided doses for 24 weeks ; or-   INTRON® A 3 MU SC TIW plus placebo matching ribavirin PO in two    divided doses for 24 weeks.

Study treatment was administered for 24 weeks. The standard INTRON® A(interferon alfa-2b, recombinant) regimen for hepatitis C wasadministered as a fixed dose of 3 MU TIW. Each patient receivedinstructions regarding the preparation and subcutaneous administrationof INTRON® A. Ribavirin was administered twice daily, morning andevening. The dose was determined by the patient's body weight at theEntry visit. Patients weighing ≦75 kg received 1000 mg daily as two 200mg capsules in the morning and three 200 mg capsules in the evening.Patients weighing >75 kg received 1200 mg daily as three 200 mg capsulesmorning and evening.

The randomization procedure was designed to balance the groups withrespect to the following Baseline characteristics:

-   pretreatment liver histology (cirrhosis or no cirrhosis);-   serum HCV-RNA/qPCR status (HCV-RNA/qPCR≦2,000,000 or    HCV-RNA/qPCR>2,000,000 copies/mL); and-   HCV Genotype (1 or other). Patients with mixed genotypes (which    include Type 1) will be classified as Type 1 for purposes of    balancing.

Efficacy

The primary efficacy objective was comparison of the two treatmentgroups with respect to-the overall response rate defined as loss ofserum HCV-RNA/qPCR at 24 weeks following the end of therapy to anundetectable level or to a level <100 copies/mL associated with animprovement in Post treatment liver biopsy as defined by the Knodell HAIinflammation score. The following secondary efficacy Endpoints were alsoexamined:

The secondary efficacy Endpoints:

-   proportions of patients with normalization of ALT at 24 weeks of    follow-up;-   proportions of patients with improvement in biopsy (Categories    I+II+III combined scores);-   changes from Baseline in the biopsy scores (Categories I+II+III    combined scores);-   response rates at Endpoint of treatment based on HCV-RNA/qPCR;-   proportion of patients with normalization of ALT at Endpoint of    treatment.-   response rates at 24 weeks of follow-up based on HCV-RNA/qPCR.

Virology: Entry Status and Change from Entry

Serum HCV-RNA/qPCR testing was performed by a central laboratory. Apositive HCV-RNA assay result was required at Baseline; only patientspositive for HCV-RNA were eligible to participate. Repeat assays 20 werescheduled at Weeks 4, 12, 24, and Follow-up Weeks 12 and 24.

Response was assessed as defined below:

Responder:

-   -   A patient was classified as a responder at a given time point if        HCV-RNA/qPCR 25 was negative (<100 copies per mL) at that time        point.

SustainedResponder:

-   -   A patient was classified as a sustained responder if the patient        was a responder at 24 weeks of follow-up. 30 Note that patients        who do not meet these criteria, including patients who        discontinued before the required HCV-RNA/qPCR evaluations were        obtained, were classified as non-responders.

OverallResponder:

-   -   Based on both serum HCV-RNA/qPCR and change in liver histology        as evaluated by the Knodell HAI Inflammation Score. A patient        was classified as an overall responder to treatment if he/she        was a sustained responder and his/her Post treatment Knodell HAI        inflammation score (sum of categories I+II+III ) improved by 2        or more units relative to the Pretreatment score.

Liver Histology

Liver biopsy was required within the six months preceding patientenrollment and at Follow-up Week 24. Evaluation of the biopsies wasperformed by a single pathologist using the Knodell Histology ActivityScore. The central pathologist was blinded with respect to patientidentification, treatment group, and the time the biopsy was obtainedrelative to treatment (Pre- or Posttreatment). Efficacy of studytreatments was assessed by comparing the degree of inflammatory activityobserved at Baseline with that present at Follow-up Week 24.

RESULTS

One hundred-ninety-five patients were enrolled at 31 internationalcenters and randomized to treatment with either INTRON® A plus ribavirin(N=98) or INTRON® A plus placebo (N=97). Three patients, two randomizedto receive INTRON® A plus ribavirin and one randomized to receiveINTRON® A plus placebo were not treated; thus, the all-treated groupsconsisted of 192 patients (96 patients each for INTRON® A plus ribavirinand INTRON® A plus placebo). Two of the three patients were not treatedbecause they did not wish to continue, the third because the protocolcriteria were not met. All discussions of efficacy and safety in thisreport are based on data for the all-treated groups.

Efficacy

The objectives of this study were to compare INTRON® A plus ribavirinwith INTRON® A plus placebo with respect to the overall response rateand the virologic response rate (based on HCV-RNA (qPCR). The primaryefficacy variable for the study is the overall response rate.

The conclusion from this regarding efficacy are as follows:

-   Combining ribavirin with INTRON® A can dramatically increase the    proportion of patients who eradicate HCV-RNA and have significant    reduction in hepatic inflammation.

The End of Follow-up overall response rate is a composite of the loss ofserum HCV-RNA(qPCR) and change in liver histology at end of follow-up(24 weeks following the end of treatment). A patient was classified asan overall responder if HCV-RNA(PCR) was negative at the 24 weekposttreatment evaluation and the posttreatment Knodell HAI inflammationscore (sum of categories I+II+III ) had improved by 2 or more unitsrelative to the pretreatment score. The End of Follow-up virologicresponse, histologic response, and overall response rates are summarizedin Tables 1, 2, and 3.

End of Follow-up HCV-RNA Response: Sustained Loss of HCV-RNA 24 WeeksFollowing the End of Treatment

The proportion of patients with eradication of HCV-RNA in the serum 24weeks following the end of treatment was tenfold greater (p<0.001) inthe group of patients treated with the combination of INTRON® A plusribavirin compared to those receiving INTRON® A monotherapy. Table 1summarizes the End of Follow-up patient response as indicated by serumHCV-RNA. TABLE 1 End of Follow-up Serum HCV-RNA: Proportion of Patientswith Eradication of HCV-RNA at 24 Weeks Following the End of Treatment.Number (%) of Patients INTRON ® A INTRON ® A Patient Response Statusplus Ribavirin plus Placebo p value All Treated 50/96 (52) 5/96 (5)<0.001 95% Confidence Interval for each treatment: 42%-62% 1%-10% fordifference between 4%-58% treatments: Responders at End of 49/80 (61)5/41 (12) Treatment^(c)

Pre- and Posttreatment biopsies were available for 81% (78/96) of thepatients treated with INTRON® A plus ribavirin and for 77% (74/96) ofthose patients who received INTRON® A plus placebo. Table 2 summarizesthe effect of treatment on hepatic inflammation for patients with bothpre- and posttreatment liver biopsy results. As with the sustained lossof HCV-RNA replication, the proportion of patients with improvement inliver inflammation was significantly greater (p<0.001) in patientsreceiving combination therapy compared to those receiving INTRON® Amonotherapy. TABLE 2 End of Follow-up Liver Histology: Improvement inLiver Histology 24 Week Following the End of Treatment Based on theKnodell HAI (I + II + III) Score. Number (%) of Patients^(b) INTRON AIINTRON A plus Ribavirin plus Placebo Patient Status (n = 78) (n = 74) pvalue^(c) Improved Biopsy^(d) 49 (51) 30 (31) <0.001a^(b)Patients with both pre- and posttreatment biopsy.^(c)Fisher's Exact test.^(d)Change from pretreatment to posttreatment in the KnodellHistological Index (HAI) score (sum of I + II + III) categorized as adecrease of 2 or more from pretreatment.

Overall Response

When the study was designed, it was recognized that because liver biopsyis an invasive procedure that it would be unlikely that posttreatmentliver biopsies would be obtained for all patients. Therefore, theprotocol and statistical analysis plan specified that the analysis foroverall response would be based on data for all treated patients andwill be estimated by a maximum likelihood method (MLE) for patientswhose overall response status could not be determined, ie, patients withnegative HCV-RNA and missing (posttreatment) biopsy evaluations. Theprotocol also specified that an additional analysis would be performedon patients with both pretreatment and posttreatment biopsy results (ie,patients with complete data). The overall response is summarized inTable 3 based on the following analyses:

-   maximum likelihood estimate (MLE);-   patients with complete data (results for both pre- and posttreatment    biopsy);

patients with missing data (either or both HCV/biopsy) treated asfailures. TABLE 3 Overall Response Rate. INTRON ® A IINTRON ® A DataAnalyzed plus Ribavirin plus Placebo p value^(b) Maximum likelihoodestimate 43% 5% <0.001 Patients with complete data^(c) 39/78 (50%) 4/74(5%) <0.001 Treat missing as failures^(d) 39/96 (41%) 4/96 (4%) <0.001a^(b)Fisher's exact test.^(c)Complete data = pre- and posttreatment biopsy results.^(d)Patients who had either virology or biopsy data missing or both werecounted as failures.

As would be anticipated from individual results for effect of treatmenton eradication of HCV-RNA at end of follow-up and improvement in hepaticinflammation, the overall response rate in the INTRON® A plus ribavirintreatment group was significantly greater (<0.001), with a 10 to 14 foldimprovement, than that observed in the INTRON® A plus placebo group forall methods of evaluation.

Logistic regression analysis was done on all Baseline demographicvariables and disease characteristics. The only Baseline statisticallysignificant characteristics predictive of End of Follow-up sustainedresponse were genotype other than 1 and viral load ≦2 million.

For number of viral copies (≦9 million, >2 million), the difference wasstatistically significant in favor of higher response rates in patientswith ≦2 million copies (Table 4).

When genotype and Baseline virus load are combined, a hierarchy ofresponse is observed. Those patients with genotype other than 1 andBaseline virus load ≦2 million copies had the best End of Follow-upresponse; those patients with genotype 1 and >2 million copies had thepoorest End of Follow-up response. TABLE 4 Disease Characteristics vsSustained Response: All-Treated Patients. Number (%) of Patients INTRONA INTRON A plus ribavirin plus Placebo Disease Characteristic^(b) (n =96) (n = 96) HCV-RNA/qPCR ≦2 million 24/36 (67)  5/29 (17) ≧2 million26/60 (43) 0/67 (0) HCV Genotype^(c) 1 16/53 (30) 2/53 (4) Other 34/43(79) 3/19 (7) Genotype/Baseline HCV-RNA/qPCR Other/≦2 million copies15/16 (93)  3/14 (21) Other/>2 million copies 18/27 (67) 0/29 (0) 1/≦2million copies  8/20 (40) 0/15 (0) 1/>2 million copies  7/33 (21) 0/38(0)a^(b)At entry, patients were stratified by number of viral copies (≦2million, >2 million), genotype (1 or other), and cirrhosis (present orabsent).

Study 2:

By basically the same methodology as described above in Study 1, asecond Study 2 was also conducted. The results are summarized below.

Efficacy

The End of Follow-up overall response rate is a composite of the loss ofserum HCV-RNA(qPCR) and change in liver histology at End of Follow-up(24 weeks following the end of treatment). A patient was classified asan overall responder if HCV-RNA(PCR) was negative at the 24 weekposttreatment evaluation and the posttreatment Knodell HAI inflammationscore (sum of categories I+II+III ) had improved by 2 or more unitsrelative to the pretreatment score. The End of Follow-up virologicresponse, histologic response, and overall response rates are summarizedin Tables 5, 6, and 7.

End of Follow-up HCV-RNA Response: Sustained Loss of HCV-RNA 24 WeeksFollowing the End of Treatment

The proportion of patients with eradication of HCV-RNA in the serum 24weeks following the end of treatment was ten-fold (p<0.001), in thegroup of patients treated with the combination of INTRON® A plusribavirin compared to those receiving INTRON® A monotherapy. Table 5summarizes the End of Follow-up patient response as indicated by serumHCV-RNA. TABLE 5 End of Follow-up Serum HCV-RNA: Proportion of Patientswith Eradication of HCV-RNA at 24 Weeks Following the End of Treatment.Number (%) of Patients INTRON A INTRON A Patient Response Status plusRibavirin plus Placebo p value All -treated Patients 34/77 (44) 3/76 (4)<0.001 95% Confidence Interval for each treatment: 33%-56% 0%-8% fordifference between 28%-52% treatments Responders at End of 34/54 (63)3/32 (9) Treatment^(c)

End of Follow-up Liver Histology: Improvement in Liver Histology 24Weeks Following the End of Treatment Based on Knodell HistologicalActivity Index (HAI) Scores (I+II+III)

Pre- and Posttreatment biopsies were available for 79% (61/77) of thepatients treated with INTRON® A plus ribavirin and for 84% (64/76) ofthose patients who received INTRON® A plus placebo. Table 6 summarizesthe effect of treatment on hepatic inflammation for patients with bothpre- and posttreatment liver biopsy results. As with the sustained lossof HCV-RNA replication, the proportion of patients with improvement inliver inflammation was significantly greater (p<0.001) in patientsreceiving combination therapy compared to those receiving INTRON® Amonotherapy. TABLE 6 End of Follow-up Liver Histology: Improvement inLiver Histology 24 Weeks Following the End of Treatment Based on theKnodell HAI (I + II + III) Score. Number (%) of Patients^(b) INTRON AINTRON A plus Ribavirin plus Placebo Patient Status (n = 61) (n = 64) pvalue^(c) Improved Biopsy^(d) 38 (49) 27 (36) <0.001a^(b)Patients with both pre-and posttreatment biopsy.^(c)Fisher's Exact test.^(d)Change from pretreatment to posttreatment in the KnodellHistological Index (HAI) score (sum of I + II + III) categorized as adecrease of 2 or more from pretreatment.

Overall Response

The overall response is summarized in Table 7 based on the followinganalyses:

-   maximum likelihood estimate (MLE);-   patients with complete data (results for both pre- and posttreatment    biopsy);

patients with missing data (either or both HCV-RNA/biopsy) treated asfailures. TABLE 7 Overall Response Rate. INTRON A INTRON A Data Analyzedplus Ribavirin plus Placebo p value^(b) ML Estimate 36.5% 2.7% <0.001Patients with complete data^(c) 25/61 (41.0%) 2/64 (3.1%) <0.001 Treatmissing as failures^(d) 25/77 (32.5%) 2/76 (2.6%) <0.001a^(b)Fisher's Exact test.^(c)Complete data = pre- and posttreatment biopsy results.^(d)Patients who had either virology or biopsy data missing or both werecounted as failures..

As would be anticipated from individual results for effect of treatmenton eradication of HCV-RNA at End of Follow-up and improvement in hepaticinflammation, the overall response rate in the INTRON A plus ribaviringroup is significantly greater (p<0.001) with a 10-14 fold improvementover that observed with INTRON A plus placebo groups for all methods ofevaluation.

Logistic regression analysis was done on all Baseline demographicvariables and disease characteristics. The only Baseline statisticallysignificant characteristic predictive of End of Follow-up sustainedresponse was genotype other than 1.

For number of viral copies (≦2 million, >2 million), there was anumerical difference in favor of higher response rates in patients with≦2 million copies (Table 8). When genotype and Baseline virus load arecombined, a hierarchy of response is observed. Those patients withgenotype other than 1 and Baseline virus load ≦2 million copies had thebest End of Follow-up response; those patients with genotype 1 and >2million copies had the poorest End of Follow-up response. TABLE 8Disease Characteristics vs Sustained Response: All-Treated Patients.Number (%) of Pataients INTRON ® A INTRON ® A plus ribavirin plusPlacebo Disease Characteristic (n = 77) (n = 76) HCV-RNA/qPCR ≦2 million 6/9 (67) 1/12 (8) >2 million 28/68 (41) 2/64 (3) HCV Genotype 1 12/46(28) 1/42 (2) Other 21/31 (68) 2/34 (6) Genotype/Baseline HCV-RNA/qPCROther/≦2 million copies   4/4 (100) 0/3 (0) Other/>2 million copies17/27 (62) 2/31 (6)  1/≦2 million copies  2/5 (40)  1/9 (11) 1/>2million copies 11/39 (28) 0/32 (0) 

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A method of treating a patient having chronic hepatitis C infectionhaving HCV genotype 1 and having a viral load of greater than 2 millioncopies/mL of serum HCV-RNA as measured by HCV-RNA/qPCR comprisingadministering to the patient a therapeutically effective combination ofribavirin and interferon-alpha for a time period of about 40 to about 50weeks, wherein the patient has fewer than about 100 copies/mL of serumHCV-RNA at the end of said period and also has less than about 100copies/mL of serum HCV-RNA for at least 24 weeks after the end of saidperiod.
 2. The method of claim 1, wherein the amount of ribavirinadministered is from about 400 to about 1200 mg per day.
 3. The methodof claim 2, wherein the amount of ribavirin administered is from about800 to about 1200 mg per day.
 4. The method of claim 1, wherein theinterferon-alpha administered is selected from interferon alpha-2a,interferon alpha-2b, a consensus interferon, a purified interferon alphaproduct or a pegylated interferon-alpha.
 5. The method of claim 2,wherein the interferon-alpha administered is selected from interferonalpha-2a, interferon alpha-2b or a purified interferon alpha product andthe amount of the interferon-alpha administered is from about 2 to about10 million international units (IU) per week on a weekly, three times aweek (EW), every other day (QOD) or daily basis.
 6. The method of claim5, wherein the amount of the interferon-alpha administered is about 3million IU TIW.
 7. The method of claim 6, wherein the interferon-alphaadministered is interferon alpha-2b.
 8. The method of claim 2, whereinthe interferon-alpha administered is consensus interferon and the amountof the interferon-alpha administered is from about 1 to about 20micrograms per week on a weekly, three times a week (TIW), every otherday (QOD) or daily basis.
 9. The method of claim 2, wherein theinterferon-alpha administered is pegylated interferon alpha-2b and theamount of the interferon-alpha administered is from about 0.5 to about2.0 micrograms/kilogram per week on a weekly, three times a week (TIW),every other day (QOD) or daily basis.
 10. The method of claim 2, whereinthe interferon-alpha administered is pegylated interferon alpha-2a andthe amount of the interferon-alpha administered is from about 20 toabout 250 micrograms/kilogram per week on a weekly, three times a week(TIW), every other day (QOD) or daily basis.
 11. The method of claim 2,wherein the patient had previously been treated with interferon-alphamonotherapy.
 12. The method of claim 3, wherein the interferon-alphaadministered is selected from interferon alpha-2a, interferon alpha-2bor a purified interferon alpha product and the amount of theinterferon-alpha administered is from about 2 to about 10 millioninternational units (IU) per week on a weekly, three times a week (TIW),every other day (QOD) or daily basis.
 13. The method of claim 12,wherein the amount of the interferon-alpha administered is about 3million IU TIW.
 14. The method of claim 13, wherein the interferon-alphaadministered is interferon alpha-2b.
 15. The method of claim 3, whereinthe interferon-alpha administered is consensus interferon and the amountof the interferon-alpha administered is from about 1 to about 20micrograms per week on a weekly, three times a week (TIW), every otherday (QOD) or daily basis.
 16. The method of claim 3, wherein theinterferon-alpha administered is pegylated interferon alpha-2b and theamount of the interferon-alpha administered is from about 0.5 to about2.0 micrograms/kilogram per week on a weekly, three times a week (TIW),every other day (QOD) or daily basis.
 17. The method of claim 3, whereinthe interferon-alpha administered is pegylated interferon alpha-2a andthe amount of the interferon-alpha administered is from about 20 toabout 250 micrograms/kilogram per week on a weekly, three times a week(TIW), every other day (QOD) or daily basis.
 18. The method of claim 3,wherein the patient had previously been treated with interferon-alphamonotherapy.